Munc13 structural transitions and oligomers that may choreograph successive stages in vesicle priming for neurotransmitter release

Abstract

How can exactly six SNARE complexes be assembled under each synaptic vesicle? Here we report cryo-EM crystal structures of the core domain of Munc13, the key chaperone that initiates SNAREpin assembly. The functional core of Munc13, consisting of C1–C2B–MUN–C2C (Munc13C) spontaneously crystallizes between phosphatidylserine-rich bilayers in two distinct conformations, each in a radically different oligomeric state. In the open conformation (state 1), Munc13C forms upright trimers that link the two bilayers, separating them by ∼21 nm. In the closed conformation, six copies of Munc13C interact to form a lateral hexamer elevated ∼14 nm above the bilayer. Open and closed conformations differ only by a rigid body rotation around a flexible hinge, which when performed cooperatively assembles Munc13 into a lateral hexamer (state 2) in which the key SNARE assembly-activating site of Munc13 is autoinhibited by its neighbor. We propose that each Munc13 in the lateral hexamer ultimately assembles a single SNAREpin, explaining how only and exactly six SNARE complexes are templated. We suggest that state 1 and state 2 may represent two successive states in the synaptic vesicle supply chain leading to “primed” ready-release vesicles in which SNAREpins are clamped and ready to release (state 3).

Ultrastructural analysis of wildtype and RIM1α knock-out active zones in a large cortical synapse

“…In addition, EDM width of SVs at 2.1-5 nm was further reduced to 14 nm in both genotypes. These findings may be related to recently described hexagonal Munc13-1 protein densities in cryo-EM (Grushin et al, 2022). The authors described a model with three states of Munc13-1 oligomers: In state 1, Munc13-1 is in the upright configuration under captured SVs of unassembled SNAREs.…”

“…In general terms the magnitude and direction of the EDM changes in our study are in accordance with this 3-state model. Furthermore, the smaller EDM width in RIM1α -/- might derive from the reduced expression of Munc13-1 at AZs (Grushin et al, 2022). An additional explanation might be the missing interaction of PIP 2 with the C 2 B domain of RIM1α.…”

Visualizing Presynaptic Active Zones and Synaptic Vesicles

“…However, larger protein displacements such as, e.g., the ones of complexin and Rab3 during SV fusion might be resolvable. Furthermore, other quantitative predictions, such as the hexameric arrangement of Munc13 below docked SVs, or the position of synaptotagmin on SVs ( Rothman et al, 2017 ; Grushin et al, 2022 ) could be experimentally tested. We should probably move from whole mounts or currently used 1 μm thick tissue to thinner sections, for these experiments.…”

“…Electron microscopy of AZs continues to provide critical insights, as for example was recently shown for structural transitions during synaptic vesicle (SV) priming ( Grushin et al, 2022 ; Lichter et al, 2022 ). Evaluating the dimensions of electron dense material below docked SVs can provide information how the chaperone Munc13, synaptotagmin, SNARE complexes, and complexins are assembled ( Rothman et al, 2017 ; Zhou et al, 2017 ).…”

Phorbolester-activated Munc13-1 and ubMunc13-2 exert opposing effects on dense-core vesicle secretion

“…Nevertheless, total and sustained secretion were reduced when Unc13b KO cells overexpressing Munc13-1 were treated with PMA, indicating that there is a negative effect of PMA x Munc13-1, which is independent of Munc13-2. The difference between Munc13-1 and ubMunc13-2, which allows the first to inhibit, but the second to stimulate, dense-core vesicle secretion when activated by phorbolester might be related to subtle differences in the length, shape or angle of the rod-like domain (Padmanarayana et al, 2021; Quade et al, 2019; Xu et al, 2017), or a difference in the ability to oligomerize (Grushin, Kalyana Sundaram, Sindelar, & Rothman, 2022), combined with the different sizes of synaptic vesicles and DVs.…”